TRP8, a transient receptor potential channel expressed in taste receptor cells

ABSTRACT

The present invention relates to the discovery, identification and characterization of a transient receptor potential channel, referred to herein as TRP8, which is expressed in taste receptor cells and associated with the perception of bitter and sweet taste. The invention encompasses TRP8 nucleotides, host cell expression systems, TRP8 proteins, fusion proteins, polypeptides and peptides, antibodies to the TRP8 protein, transgenic animals that express a TRP8 transgene, and recombinant “knock-out” animals that do not express TRP8. The invention further relates to methods for identifying modulators of the TRP8-mediated taste response and the use of such modulators to either inhibit or promote the perception of bitterness or sweetness. The modulators of TRP8 activity may be used as flavor enhancers in foods, beverages and pharmaceuticals.

1. INTRODUCTION

[0001] The present invention relates to the discovery, identificationand characterization of a transient receptor potential channel, referredto herein as TRP8, which is expressed in taste receptor cells andassociated with the perception of bitter and sweet taste. The inventionencompasses TRP8 nucleotides, host cell expression systems, TRP8proteins, fusion proteins, polypeptides and peptides, antibodies to theTRP8 protein, transgenic animals that express a TRP8 transgene, andrecombinant “knock-out” animals that do not express TRP8. The inventionfurther relates to methods for identifying modulators of theTRP8-mediated taste response and the use of such modulators to eitherinhibit or promote the perception of bitterness or sweetness. Themodulators of TRP8 activity may be used as flavor enhancers in foods,beverages and pharmaceuticals.

2. BACKGROUND OF THE INVENTION

[0002] Mammals are generally thought to have five basic categories oftaste perception: salt, sour, sweet, bitter and umami (monosodiumglutamate) (for review, see Lindemann, 1996, Physiological Reviews,76:719-766; Herness and Gilbertson, 1999, Annu Rev. Physiol.,61:873:900). The taste signals are sensed by specialized taste receptorcells (TRCs), which are organized into taste buds. Each taste budcomprises between about 50 and 100 individual cells grouped into acluster that is between 20 and 40 microns in diameter. Nerve fibersenter from the base of the taste bud and synapse onto some of the tastereceptor cells. Typically, a single TRC contacts several sensory nervefibers, and each sensory fiber innervates several TRCs in the same tastebud (Lindemann, supra).

[0003] TRCs of most, if not all, vertebrate species possessvoltage-gated sodium, potassium, and calcium ion channels withproperties similar to those of neurons (Kinnamon, S. C. & Margolskee, R.F., 1996, Curr. Opin. Neurobiol. 6:506-513). Different types of primarytastes appear to utilize different types of transduction mechanisms, andcertain types of tastes may employ multiple mechanisms which may reflectvarying nutritional requirements amongst species (Kinnamon & Margolskee,supra).

[0004] Bitter and sweet taste transduction are thought to involve cAMPand IP₃ (Kinnamon & Margolskee, supra). The bitter compound denatoniumcauses calcium ion release from rat TRCs and the rapid elevation of IP₃levels in rodent taste tissue (Id., citing Bernhardt, S J. et al., 1996,J. Physiol. (London) 490:325-336 and Akabas, M. H., et al., 1988,Science 242:1047-1050). Since denatonium cannot pass the cell membrane,it has been suggested that it may activate G-protein-coupled receptors,whereby the α and/or βγ G protein subunits would activate phospholipaseC, leading to IP₃ generation and the release of calcium ions (Kinnamon &Margolskee, supra).

[0005] In recent years, a taste-specific G protein termed “gustducin”,which is homologous to the retinal G protein, transducin, has beencloned and characterized (Id., citing McLaughlin, S. et al., 1992,Nature (London) 357:563-569). It is believed that gustducin plays adirect role in both bitter and sweet transduction. For example,gustducin and subunit (∝-gustducin) null (knockout) mice had a reducedaversion to bitter compounds. Unexpectedly, the mice also exhibited apreference for sweet compounds suggesting involvement of gustducin insweet transduction.

[0006] Recent biochemical experiments have demonstrated that tastereceptor preparations activate transducin and gustducin in response todenatonium and other bitter compounds (Ming et al., 1998, Proc. Natl.Acad. Sci. USA 95:8933-8).

[0007] To thoroughly understand the molecular mechanisms underlyingtaste sensation, it is important to identify each molecular component inthe taste signal transduction pathways. The present invention relates tothe cloning of an ion channel, TRP8 (transient receptor potentialchannel 8), that is believed to be involved in taste transduction andmay be involved in the changes in intra-cellular calcium ions associatedwith bitter taste perception.

3. SUMMARY OF THE INVENTION

[0008] The present invention relates to the discovery, identificationand characterization of a transient receptor potential (TRP) channel,referred to hereafter as TRP8, that participates in the taste signaltransduction pathway. TRP8 is a channel protein with a high degree ofstructural similarity to the family of calcium channel proteins known astransient receptor potential channels. As demonstrated by Northern Blotanalysis, expression of the TRP8 transcript is tightly regulated, withthe highest level of gene expression found in taste tissue, moderateexpression in stomach and small intestine, and very low level expressionin uterus and testis. In situ hybridization indicated expression of TRP8in circumvallate and foliate papillae, but not in the surroundingnon-gustatory epithelia. Additionally, the general pattern of TRP8expression was comparable to that of α-gustducin, although theα-gustducin signal was somewhat more intense.

[0009] The present invention encompasses TRP8 nucleotides, host cellsexpressing such nucleotides and the expression products of suchnucleotides. The invention encompasses TRP8 protein, TRP8 fusionproteins, antibodies to the TRP8 channel protein and transgenic animalsthat express a TRP8 transgene or recombinant knock-out animals that donot express the TRP8 protein.

[0010] Further, the present invention also relates to screening methodsthat utilize the TRP8 gene and/or TRP8 gene products as targets for theidentification of compounds which modulate, i.e., act as agonists orantagonists, of TRP8 activity and/or expression. Compounds whichstimulate taste responses similar to those of bitter tastants can beused as additives to provoke a desired aversive response—for example todiscourage ingestion of compositions containing these compounds bychildren or animals. Compounds which inhibit the activity of the TRP8channel may be used to block the perception of bitterness. Theinhibitors of TRP8 may be used as flavor enhancers in foods, beveragesor pharmaceuticals by decreasing or eliminating the perception of bittertaste.

[0011] The invention is based, in part, on the discovery of a channelprotein expressed at high levels in taste receptor cells. In tastetransduction, bitter compounds are thought to act via the G-proteins,such as gustducin, which in turn regulate second messenger systems.Co-localization of α-gustducin, γ-gustducin, phospholipase Cβ₂ (PLCβ₂)and TRP8 to one subset of taste receptor cells indicates that they mayfunction in the same transduction pathway. It is believed that TRP8responds to tastant induced inositol triphosphate (IP₃)/diacylglycerol(DAG) generation by flooding the taste cell with extracellular calciumand activating calcium dependent down stream messengers leading totransmitter release into the synapse and activation of afferentgustatory nerves.

3.1. DEFINITIONS

[0012] As used herein, italicizing the name of TRP8 shall indicate theTRP8 gene, in contrast to its encoded protein product which is indicatedby the name of TRP8 in the absence of italicizing. For example, “TRP8”shall mean the TRP8 gene, whereas “TRP8” shall indicate the proteinproduct of the TRP8 gene.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1. Nucleotide sequence of the murine TRP8 cDNA encodingmurine TRP8.

[0014]FIG. 2. Deduced amino acid sequence of the murine TRP8 transientreceptor potential channel.

[0015]FIG. 3A-B. Nucleotide sequence of the human TRP8 cDNA encodinghuman TRP8.

[0016]FIG. 4. Deduced amino acid sequence of the human TRP8 proteintransient receptor potential channel.

[0017]FIG. 5. Amino acid sequence of the murine TRP8 (upper lines);versus human TRP8 (lower lines). Each pair of lines corresponds to apredicted mouse/human exon.

[0018] FIGS. 6A-C. Predicted topography of the TRP8 protein transientreceptor potential channel in the membrane.

[0019]FIG. 7. Distribution of TRP8 MRNA and protein in mouse tissues.(a) Autoradiogram of a northern blot hybridized with a TRP8 cDNA probe.Each lane contained 25 μg total RNA isolated from the following mousetissues: circumvallate and foliate papillae-enriched taste tissue (Tastetissue), lingual tissue devoid of taste buds (Non-taste), brain, retina,olfactory epithelium (Olf. Epi.), stomach, small intestine (Small Int.),thymus, heart, lung, spleen, skeletal muscle (Skele. Mus.), liver,kidney, uterus and testis. A 4.5 kb transcript was detected in tastetissue, stomach and small intestine, and to a much lesser extent, inuterus and testis. To control for MRNA quantity the same blot wasstripped and reprobed with a β-actin cDNA probe (lower panel). The sizein kilobases (kb) of RNA markers is indicated at the right-hand side.(b) Autoradiogram of a western blot probed with an anti-TRP8 antibody.Protein extracts (50 μg) prepared from the murine tissues indicated wereelectrophoresed, transferred to a nitrocellulose membrane, then the blotincubated with an antibody against the carboxyl-terminal of TRP8.Immunoreactive protein of ˜130 kD, the predicted molecular weight ofTRP8, was detected in stomach and small intestine; a higher molecularweight species was identified in liver and kidney. Molecular sizemarkers are given in kilodaltons.

[0020]FIG. 8. TRP8 mRNA is expressed in taste receptor cells. Sectionsof murine lingual epithelia containing circumvallate and foliatepapillae were hybridized with ³³P-labeled antisense RNA probes for TRP8(a,c) and α-gustducin (d), and subjected to autoradiography.Photomicrographs of circumvallate (a) and foliate (b) papillaehybridized to the antisense TRP8 probe demonstrates expression of TRP8in a subset of TRCs. (d) Shows hybridization of an α-gustducin antisenseprobe to foliate papillae. Hybridization controls with sense probesshowed the absence of non-specific binding of the TRP8 probe (b) or theα-Gustducin probe (e).

[0021]FIG. 9. Co-localization in TRCs of TRP8 and other signaltransduction elements. Immunofluorescence of Gγ13 (a) and TRP8 (b) inthe same longitudinal section of mouse taste papillae section: (c) isthe overlay of a and b. Immunofluorescence of TRP8 (d) and α-gustducin(e) in the same section: (f) is the overlay of d and e.Immunofluorescence of PLCβ2 (g) and TRP8 (h) in the same section: (i) isthe overlay of g and h.

[0022]FIG. 10. Profiling the pattern of expression of TRP8, a-gustducin,Gβ1, Gβ3, Gγ13 and PLCβ2 in taste tissue and taste cells. Left panel:Southern hybridization to RT-PCR products from murine taste tissue (T)and control non-taste lingual tissue (N). 3′-region probes from TRP8,α-gustducin (Gust), Gβ1, Gβ3, Gγ13, PLCβ2 and glyceraldehyde 3-phosphatedehydrogenase (G3PDH) were used to probe the blots. Note that TRP8,α-gustducin, Gβ1, Gβ3, Gγ13 and PLCβ2 were all expressed in tastetissue, but not in non-taste tissue. Right panel: Southern hybridizationto RT-PCR products from 24 individually amplified taste receptor cellsfrom a transgenic mouse expressing green fluorescent protein (GFP) fromthe gustducin promoter. 19 cells were GFP-positive (+), 5 cells wereGFP-negative (−). Expression of TRP8, α-gustducin, Gβ3, Gγ13and PLCβ2was fully coincident. 15 of 19 TRP8-positive cells were also positivefor Gβ1. G3PDH served as a positive control to demonstrate successfulamplification of products.

[0023]FIG. 11. TRP8, but not mTrp 1-7, is detected by PCR in tastetissue. PCR amplifications of TRP8 and mTrp 1-7 were performed usingnon-degenerate primers specific for each Trp family member. Taste cDNA(upper panels) and brain cDNA (lower panels) provided templates foramplification. Amplified material was resolved in a 1.2% agarose gel.Bands of the expected molecular weight were sequenced to verify theidentity of the Trp channel amplified. Positive (G3PDH primers) andnegative (no primers) controls are shown (right panels).

[0024]FIG. 12. Heterologous expression of TRP8. Xenopus oocytes wereinjected with 50 ng of TRP8 cRNA (a) or 50 nl of water (b); two daysafter injection, oocytes were treated with thapsigargin (2 μM), followedby the addition of Ca++(10 mM) or EGTA as indicated (arrows). The tracesrepresent currents induced at negative membrane potentials (commandvoltage −80 mV). (c) I-V curve for oocytes injected with TRP8 cRNA orwater demonstrates a reversal potential, consistent with Ca++ activationof the endogenous calcium-activated chloride conductance (ICl_(Ca)). (d)The maximal inward current elicited with external Ca++ present in thebathing media for oocytes injected with TRP8 cRNA or water (control).

[0025]FIG. 13. TRP8 functions as a Ca++ channel. Xenopus oocytes wereinjected with 50 ng of TRP8 cRNA (right panels) or 50 nl of water (leftpanels); two days after injection, oocytes were treated withthapsigargin (2 μM), followed by the addition of Ca++ (10 mM).

[0026]FIG. 14. Potential signal transduction pathways in TRCs utilizingTRP8. Responses to bitter compounds such as denatonium are initiated bybinding to one or more gustducin-coupled receptors of the T2R/TBRfamily. Activation of the gustducin heterotrimer releases its βγmoiety(e.g. Gβ3/Gγ13) which stimulates PLCβ2, resulting in production of IP₃and DAG. IP₃ binds to its receptors e.g. IP₃R3 and causes the release ofCa⁺⁺ from intracellular stores, triggering activation of TRP8 channels,which ultimately leads to the influx of Ca⁺⁺ through TRP8 channels. DAGmay act directly on TRP8 to lead to Ca⁺⁺ influx. Artificial sweetenersmay depend on a similar transduction pathway, but with sweet-responsivereceptors, e.g., T1R3 coupled to gustducin or other G proteinsinitiating the signal that leads to the production of IP₃ and DAG andstimulation of TRP8.

5. DETAILED DESCRIPTION OF THE INVENTION

[0027] TRP8 is a channel protein that participates in receptor-mediatedtaste signal transduction and belongs to the family of calcium channelproteins known as transient receptor potential channels (Montell C.,1997, Mol. Pharmacol. 52:755-763) The present invention encompasses TRP8nucleotides, TRP8 proteins and peptides, as well as antibodies to theTRP8 protein. The invention also relates to host cells and animalsgenetically engineered to express the TRP8 channel or to inhibit or“knock-out” expression of the animal's endogenous TRP8.

[0028] The invention further provides screening assays designed for theidentification of modulators, such as agonists and antagonists, of TRP8activity. The use of host cells that naturally express TRP8 orgenetically engineered host cells and/or animals offers an advantage inthat such systems allow the identification of compounds that affect thesignal transduced by the TRP8 protein.

[0029] Various aspects of the invention are described in greater detailin the subsections below.

5.1. THE TRP8 GENE

[0030] The cDNA sequence and deduced amino acid sequence of murine TRP8are shown in FIGS. 1 and 2, respectively. The cDNA and deduced aminoacid sequence of human TRP8 are shown in FIGS. 3 and 4, respectively.

[0031] The TRP8 nucleotide sequences of the invention include: (a) theDNA sequences shown in FIG. 1 or 3 or contained in the cDNA clone pMR24within E. coli strain XL10 Gold as deposited with the American TypeCulture Collection (ATCC Accession No. ); (b) nucleotide sequences thatencode the amino acid sequence shown in FIG. 2 or 4 or the TRP8 aminoacid sequence encoded by the cDNA clone pMR24 as deposited with theATCC; (c) any nucleotide sequence that (i) hybridizes to the nucleotidesequence set forth in (a) or (b) under stringent conditions, e.g.,hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols in MolecularBiology, Vol. I, Green Publishing Associates, Inc., and John Wiley &sons, Inc., New York, at p. 2.10.3) and (ii) encodes a functionallyequivalent gene product; and (d) any nucleotide sequence that hybridizesto a DNA sequence that encodes the amino acid sequence shown in FIG. 1or 3, or that is contained in cDNA clone pMR24 as deposited with theATCC, under less stringent conditions, such as moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989 supra), yet which still encodes a functionally equivalent TRP8 geneproduct. Functional equivalents of the TRP8 protein include naturallyoccurring TRP8 present in species other than mice and humans. Theinvention also includes degenerate variants of sequences (a) through(d). The invention also includes nucleic acid molecules, that may encodeor act as TRP8 antisense molecules, useful, for example, in TRP8 generegulation (for and/or as antisense primers in amplification reactionsof TRP8 gene nucleic acid sequences).

[0032] In addition to the TRP8 nucleotide sequences described above,homologs of the TRP8 gene present in other species can be identified andreadily isolated, without undue experimentation, by molecular biologicaltechniques well known in the art. For example, cDNA libraries, orgenomic DNA libraries derived from the organism of interest can bescreened by hybridization using the nucleotides described herein ashybridization or amplification probes.

[0033] The invention also encompasses nucleotide sequences that encodemutant TRP8s, peptide fragments of the TRP8, truncated TRP8, and TRP8fusion proteins. These include, but are not limited to nucleotidesequences encoding polypeptides or peptides corresponding to the TM(transmembrane) and/or CD (cytoplasmic) domains of TRP8 or portions ofthese domains; truncated TRP8s in which one or two of the domains isdeleted, e.g., a functional TRP8 lacking all or a portion of the CDregion. Certain of these truncated or mutant TRP8 proteins may act asdominant-negative inhibitors of the native TRP8 protein. Nucleotidesencoding fusion proteins may include but are not limited to full lengthTRP8, truncated TRP8 or peptide fragments of TRP8 fused to an unrelatedprotein or peptide such as an enzyme, fluorescent protein, luminescentprotein, etc., which can be used as a marker.

[0034] TRP8 nucleotide sequences may be isolated using a variety ofdifferent methods known to those skilled in the art. For example, a cDNAlibrary constructed using RNA from a tissue known to express TRP8 can bescreened using a labeled TRP8 probe. Alternatively, a genomic librarymay be screened to derive nucleic acid molecules encoding the TRP8channel protein. Further, TRP8 nucleic acid sequences may be derived byperforming PCR using two oligonucleotide primers designed on the basisof the TRP8 nucleotide sequences disclosed herein. The template for thereaction may be cDNA obtained by reverse transcription of mRNA preparedfrom cell lines or tissue known to express TRP8.

[0035] The invention also encompasses (a) DNA vectors that contain anyof the foregoing TRP8 sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingTRP8 sequences operatively associated with a regulatory element thatdirects the expression of the TRP8 coding sequences; (c) geneticallyengineered host cells that contain any of the foregoing TRP8 sequencesoperatively associated with a regulatory element that directs theexpression of the TRP8 coding sequences in the host cell; and (d)transgenic mice or other organisms that contain any of the foregoingTRP8 sequences. As used herein, regulatory elements include but are notlimited to inducible and non-inducible promoters, enhancers, operatorsand other elements known to those skilled in the art that drive andregulate expression.

5.2. TRP8 PROTEINS AND POLYPEPTIDES

[0036] TRP8 protein, polypeptides and peptide fragments, mutated,truncated or deleted forms of the TRP8 and/or TRP8 fusion proteins canbe prepared for a variety of uses, including but not limited to thegeneration of antibodies, the identification of other cellular geneproducts involved in the regulation of TRP8 mediated taste perception,and the screening for compounds that can be used to modulate tasteperception such as bitter blocking agents and taste modifiers.

[0037]FIGS. 2 and 4 show the deduced amino acid sequence of the murineand human TRP8 protein, respectively. The TRP8 amino acid sequences ofthe invention include the amino acid sequence shown in FIG. 2 or FIG. 4,or the amino acid sequence encoded by cDNA clone pMR24 as deposited withthe ATCC. Further, TRP8s of other species are encompassed by theinvention. In fact, any TRP8 protein encoded by the TRP8 nucleotidesequences described in Section 5.1, above, is within the scope of theinvention.

[0038] The invention also encompasses proteins that are functionallyequivalent to the TRP8 encoded by the nucleotide sequences described inSection 5.1, as judged by any of a number of criteria, including but notlimited to the ability of a bitter tastant to trigger the influx ofcalcium from extracellular calcium stores into a taste receptor cellexpressing said protein, leading to transmitter release from the tastereceptor cell into the synapse and activation of an afferent nerve. Suchfunctionally equivalent TRP8 proteins include but are not limited toproteins having additions or substitutions of amino acid residues withinthe amino acid sequence encoded by the TRP8 nucleotide sequencesdescribed, above, in Section 5.1, but which result in a silent change,thus producing a functionally equivalent gene product.

[0039] Peptides corresponding to one or more domains of TRP8 (e.g.,transmembrane (TM) or cellular domain (CD)), truncated or deleted TRP8s(e.g., TRP8 in which the TM and/or CD is deleted) as well as fusionproteins in which the full length TRP8, a TRP8 peptide or a truncatedTRP8 is fused to an unrelated protein are also within the scope of theinvention and can be designed on the basis of the TRP8 nucleotide andTRP8 amino acid sequences disclosed herein. Such fusion proteins includefusions to an enzyme, fluorescent protein, or luminescent protein whichprovide a marker function.

[0040] While the TRP8 polypeptides and peptides can be chemicallysynthesized (e.g, see Creighton, 1983, Proteins: Structures andMolecular Principles, W. H. Freeman & Co., N.Y.), large polypeptidesderived from TRP8 and the full length TRP8 itself may be advantageouslyproduced by recombinant DNA technology using techniques well known inthe art for expressing a nucleic acid containing TRP8 gene sequencesand/or coding sequences. Such methods can be used to constructexpression vectors containing the TRP8 nucleotide sequences described inSection 5.1 and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.(See, for example, the techniques described in Sambrook et al., 1989,supra, and Ausubel et al., 1989, supra).

[0041] A variety of host-expression vector systems may be utilized toexpress the TRP8 nucleotide sequences of the invention. Where the TRP8peptide or polypeptide is expressed as a soluble derivative (e.g.,peptides corresponding to TM and/or CD) and is not secreted, the peptideor polypeptide can be recovered from the host cell. Alternatively, wherethe TRP8 peptide or polypeptide is secreted the peptide or polypeptidesmay be recovered from the culture media. However, the expression systemsalso include engineered host cells that express TRP8 or functionalequivalents, anchored in the cell membrane. Purification or enrichmentof the TRP8 from such expression systems can be accomplished usingappropriate detergents and lipid micelles and methods well known tothose skilled in the art. Such engineered host cells themselves may beused in situations where it is important not only to retain thestructural and functional characteristics of the TRP8, but to assessbiological activity, i.e., in drug screening assays.

[0042] The expression systems that may be used for purposes of theinvention include but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors containing TRP8 nucleotide sequences; yeasttransformed with recombinant yeast expression vectors containing TRP8nucleotide sequences or mammalian cell systems harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells or from mammalian viruses.

[0043] Appropriate expression systems can be chosen to ensure that thecorrect modification, processing and sub-cellular localization of theTRP8 channel protein occurs. To this end, eukaryotic host cells whichpossess the ability to properly modify and process the TRP8 channelprotein are preferred. For long-term, high yield production ofrecombinant TRP8 channel protein, such as that desired for developmentof cell lines for screening purposes, stable expression is preferred.Rather than using expression vectors which contain origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements and a selectable marker gene,i.e., tk, hgprt, dhfr, neo, and hygro gene, to name a few. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in enriched media, and then switched to a selective media.Such engineered cell lines may be particularly useful in screening andevaluation of compounds that modulate the endogenous activity of theTRP8 gene product.

5.3. TRANSGENIC ANIMALS

[0044] The TRP8 gene products can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-humanprimates, e., baboons, monkeys, and chimpanzees may be used to generateTRP8 transgenic animals.

[0045] Any technique known in the art may be used to introduce the TRP8transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell,56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

[0046] The present invention provides for transgenic animals that carrythe TRP8 transgene in all their cells, as well as animals which carrythe transgene in some, but not all their cells, i.e., mosaic animals.The transgene may also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal., (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236).The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art. When it is desired thatthe TRP8 transgene be integrated into the chromosomal site of theendogenous TRP8 gene, gene targeting is preferred. Briefly, when such atechnique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous TRP8 gene are designed for thepurpose of integrating, via homologous recombination with chromosomalsequences, into and disrupting the function of the nucleotide sequenceof the endogenous TRP8 gene.

[0047] Once transgenic animals have been generated, the expression ofthe recombinant TRP8 gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of TRP8 gene-expressing tissue may also beevaluated immunocytochemically using antibodies specific for the TRP8transgene product.

5.4. ANTIBODIES TO TRP8 PROTEINS

[0048] Antibodies that specifically recognize one or more epitopes ofTRP8, or epitopes of conserved variants of TRP8, or peptide fragments ofTRP8 are also encompassed by the invention. Such antibodies include butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0049] The antibodies of the invention may be used, for example, inconjunction with compound screening schemes, as described, below, inSection 5.5, for the evaluation of the effect of test compounds onexpression and/or activity of the TRP8 gene product.

[0050] For production of antibodies, various host animals may beimmunized by injection with a TRP8 protein, or TRP8 peptide. Such hostanimals may include but are not limited to rabbits, mice, and rats, toname but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG-(Bacille Calmette-Guerin) and Corynebacterium parvum.

[0051] Polyclonal antibodies comprising heterogeneous populations ofantibody molecules, may be derived from the sera of the immunizedanimals. Monoclonal antibodies may be obtained by any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S.Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor etal., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad.Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al.,1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-9). Such antibodies may be of any immunoglobulin class including IgG,IgM, IgE, IgA, IgD and any subclasses thereof. The hybridoma producingthe mAb of this invention may be cultivated in vitro or in vivo.Production of high titres of Mabs in vivo makes this the presentlypreferred method of production.

[0052] In addition, techniques developed for the production of “chimericantibodies” by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used(Morrison et al., 1984, Proc. Nat'l. Acad. Sci., 81:6851-6855; Neubergeret al., 1984, Nature, 312: 604-608; Takeda et al. 1985, Nature 314:452-454). Alternatively, techniques developed for the production ofhumanized antibodies (U.S. Pat. No. 5,585,089) or single chainantibodies (U.S. Pat. No. 4,946,778 Bird, 1988, Science 242: 423-426;Huston et al., 1988, Proc. Nat'l. Acad. Sci USA, 85: 5879-5883; and Wardet al., 1989, Nature 334: 544-546) may be used to produce antibodiesthat specifically recognize one or more epitopes of TRP8.

5.5. SCREENING ASSAYS FOR DRUGS AND OTHER CHEMICAL COMPOUNDS USEFUL INREGULATION OF TASTE PERCEPTION

[0053] The present invention relates to screening assay systems designedto identify compounds or compositions that modulate TRP8 activity orTRP8 gene expression, and thus, may be useful for modulation of bittertaste perception.

[0054] In accordance with the invention, a cell-based assay system canbe used to screen for compounds that modulate the activity of the TRP8and thereby, modulate the perception of bitterness. To this end, cellsthat endogenously express TRP8 can be used to screen for compounds.Alternatively, cell lines, such as 293 cells, COS cells, CHO cells,fibroblasts, and the like, genetically engineered to express TRP8 can beused for screening purposes. Preferably, host cells geneticallyengineered to express a functional TRP8 are those that respond toactivation by bitter tastants, such as taste receptor cells. Further,ooyctes or liposomes engineered to express the TRP8 channel protein maybe used in assays developed to identify modulators of TRP8 activity.

[0055] The present invention provides for methods for identifying acompound that induces the perception of a bitter taste (a “bitternessactivator”) comprising (i) contacting a cell expressing the TRP8 channelprotein with a test compound and measuring the level of TRP8 activation;(ii) in a separate experiment, contacting a cell expressing the TRP8channel protein with a vehicle control and measuring the level of TRP8activation where the conditions are essentially the same as in part (i),and then (iii) comparing the level of activation of TRP8 measured inpart (i) with the level of activation of TRP8 in part (ii), wherein anincreased level of activated TRP8 in the presence of the test compoundindicates that the test compound is a TRP8 activator.

[0056] The present invention also provides for methods for identifying acompound that inhibits the perception of a bitter taste (a “bitternessinhibitor”) comprising (i) contacting a cell expressing the TRP8 channelprotein with a test compound in the presence of a bitter tastant andmeasuring the level of TRP8 activation; (ii) in a separate experiment,contacting a cell expressing the TRP8 channel protein with a bittertastant and measuring the level of TRP8 activation, where the conditionsare essentially the same as in part (i) and then (iii) comparing thelevel of activation of TRP8 measured in part (i) with the level ofactivation of TRP8 in part (ii), wherein a decrease level of activationof TRP8 in the presence of the test compound indicates that the testcompound is a TRP8 inhibitor.

[0057] A “bitter tastant”, as defined herein, is a compound or molecularcomplex that induces, in a subject, the perception of a bitter taste. Inparticular, a bitter tastant is one which results in the activation ofthe TRP8 channel protein resulting in an influx of Ca⁺² into the cell.Examples of bitter tastants include but are not limited to denatoniumbenzoate (“denatonium”; also “DEN”), quinine hydrochloride (“quinine”;also “QUI”), strychnine hydrochloride (“strychnine”; also “STR”),nicotine hemisulfate (“nicotine”; also “NIC”), atropine hydrochloride(“atropine”; also “ATR”), sparteine, naringin, caffeic acid(“caffeine”;also “CAF”), quinacrine, and epicatechin. See Ming et al., 1999, Proc.Natl. Acad. Sci. U.S.A. 96:9903-9908, incorporated by reference herein.

[0058] In utilizing such cell systems, the cells expressing the TRP8channel protein are exposed to a test compound or to vehicle controls(e.g., placebos). After exposure, the cells can be assayed to measurethe expression and/or activity of components of the signal transductionpathway of TRP8, or the activity of the signal transduction pathwayitself can be assayed.

[0059] The ability of a test molecule to modulate the activity of TRP8may be measured using standard biochemical and physiological techniques.Responses such as activation or suppression of catalytic activity,phosphorylation or dephosphorylation of TRP8 and/or other proteins,activation or modulation of second messenger production, changes incellular ion levels, association, dissociation or translocation ofsignaling molecules, or transcription or translation of specific genesmay be monitored. In non-limiting embodiments of the invention, changesin intracellular Ca²⁺ levels may be monitored by the fluorescence ofindicator dyes such as indo, fura, etc. In addition activation of cyclicnucleotide phosphodiesterase, adenylate cyclase, phospholipases ATPasesand Ca²⁺ sensitive release of neurotransmitters may be measured toidentify compounds that modulate TRP8 signal transduction. Further,changes in membrane potential resulting from modulation of the TRP8channel protein can be measured using a voltage clamp or patch recordingmethods.

[0060] For example, after exposure to a test compound, cell lysates canbe assayed for increased intracellular levels of Ca²⁺ and activation ofcalcium dependent down stream messengers such as phosphodiesterase,phospholipases, ATPases or cAMP. The ability of a test compound toincrease intracellular levels of Ca²⁺ and activate phosphodiesterase ordecrease cAMP levels compared to those levels seen with cells treatedwith a vehicle control, indicates that the test compound acts as anagonist (i.e., is a TRP8 activator) and induces signal transductionmediated by the TRP8 expressed by the host cell. The ability of a testcompound to inhibit bitter tastant induced calcium influx and inhibitphosphodiesterase or increase cAMP levels compared to those levels seenwith a vehicle control indicates that the test compound acts as anantagonist (i.e., is a TRP8 inhibitor) and inhibits signal transductionmediated by TRP8.

[0061] In a specific embodiment of the invention, levels of cAMP can bemeasured using constructs containing the cAMP responsive element linkedto any of a variety of different reporter genes. Such reporter genes mayinclude but are not limited to chloramphenicol acetyltransferase (CAT),luciferase, β-glucuronidase (GUS), growth hormone, or placental alkalinephosphatase (SEAP). Such constructs are introduced into cells expressingTRP8 channel protein thereby providing a recombinant cell useful forscreening assays designed to identify modulators of TRP8 activity.

[0062] Following exposure of the cells to the test compound, the levelof reporter gene expression may be quantitated to determine the testcompound's ability to regulate TRP8 activity. Alkaline phosphataseassays are particularly useful in the practice of the invention as theenzyme is secreted from the cell. Therefore, tissue culture supernatantmay be assayed for secreted alkaline phosphatase. In addition, alkalinephosphatase activity may be measured by calorimetric, bioluminescent orchemilumenscent assays such as those described in Bronstein, I. et al.(1994, Biotechniques 17: 172-177). Such assays provide a simple,sensitive easily automatable detection system for pharmaceuticalscreening.

[0063] Additionally, to determine intracellular cAMP concentrations, ascintillation proximity assay (SPA) may be utilized (SPA kit is providedby Amersham Life Sciences, Illinois). The assay utilizes ¹²⁵I-labelcAMP, an anti-cAMP antibody, and a scintillant-incorporated microspherecoated with a secondary antibody. When brought into close proximity tothe microsphere through the labeled cAMP-antibody complex, ¹²⁵I willexcite the scintillant to emit light. Unlabeled cAMP extracted fromcells competes with the ¹²⁵I-labeled cAMP for binding to the antibodyand thereby diminishes scintillation. The assay may be performed in96-well plates to enable high-throughput screening and 96 well-basedscintillation counting instruments such as those manufactured by Wallacor Packard may be used for readout.

[0064] In yet another embodiment of the invention, levels ofintracellular Ca²⁺ can be monitored using Ca²⁺ indication dyes, such asFluo-3 and Fura-Red using methods such as those described in Komuro andRakic, 1998, In: The Neuron in Tissue Culture. L. W. Haymes, Ed. Wiley,New York.

[0065] Test activators which activate the activity of TRP8, identifiedby any of the above methods, may be subjected to further testing toconfirm their ability to induce a bitterness perception. Test inhibitorswhich inhibit the activation of TRP8 by bitter tastants, identified byany of the above methods, may then be subjected to further testing toconfirm their inhibitory activity. The ability of the test compound tomodulate the activity of the TRP8 receptor may be evaluated bybehavioral, physiologic, or in vitro methods.

[0066] For example, a behavioral study may be performed where a testanimal may be offered the choice of consuming a composition comprisingthe putative TRP8 inhibitor and the same composition without the addedcompound. A preference for the composition comprising a test compound,indicated, for example, by greater consumption, would have a positivecorrelation with TRP8 inhibitory activity. Additionally, avoidance by atest animal of food containing a putative activator of TRP8 would have apositive correlation with the identification of an bitterness activator.

[0067] In addition to cell based assays, non-cell based assay systemsmay be used to identify compounds that interact with, e.g., bind toTRP8. Such compounds may act as antagonists or agonists of TRP8 activityand may be used to regulate bitter taste perception.

[0068] To this end, soluble TRP8 may be recombinantly expressed andutilized in non-cell based assays to identify compounds that bind toTRP8. The recombinantly expressed TRP8 polypeptides or fusion proteinscontaining one or more of the domains of TRP8 prepared as described inSection 5.2, infra, can be used in the non-cell based screening assays.For example, peptides corresponding to one or more of the cytoplasmic ortransmembrane domains of TRP8, or fusion proteins containing one or moreof the cytoplasmic or transmembrane domains of TRP8 can be used innon-cell based assay systems to identify compounds that bind to thecytoplasmic portion of the TRP8; such compounds may be useful tomodulate the signal transduction pathway of the TRP8. In non-cell basedassays the recombinantly expressed TRP8 may be attached to a solidsubstrate such as a test tube, microtitre well or a column, by meanswell known to those in the art (see Ausubel et al., ura). The testcompounds are then assayed for their ability to bind to the TRP8.

[0069] The TRP8 channel protein may be one which has been fully orpartially isolated from other molecules, or which may be present as partof a crude or semi-purified extract. As a non-limiting example, the TRP8channel protein may be present in a preparation of taste receptor cellmembranes. In particular embodiments of the invention, such tastereceptor cell membranes may be prepared as set forth in Ming, D. et al.,1998, Proc. Natl. Sci. U.S.A. 95:8933-8938, incorporated by referenceherein. Specifically, bovine circumvallate papillae (“taste tissue”,containing taste receptor cells), may be hand dissected, frozen inliquid nitrogen, and stored at −80° C. prior to use. The collectedtissues may then be homogenized with a Polytron homogenizer (threecycles of 20 seconds each at 25,000 RPM) in a buffer containing 10 mMTris at pH 7.5, 10% vol/vol glycerol, 1 mM EDTA, 1 mM DTT, 10 μg/μlpepstatin A, 10 μg/μl leupeptin, 10 μg/μl aprotinin, and 100 μM4-(2-amino ethyl) benzenesulfoyl fluoride hydrochloride. Afterparticulate removal by centrifugation at 1,500×g for 10 minutes, tastemembranes may be collected by centrifugation at 45,000×g for 60 minutes.The pelleted membranes may then be rinsed twice, re-suspended inhomogenization buffer lacking protease inhibitors, and furtherhomogenized by 20 passages through a 25 gauge needle. Aliquots may thenbe either flash frozen or stored on ice until use. As anothernon-limiting example, the taste receptor may be derived from recombinantclones (see Hoon, M. R. et al., 1999 Cell 96, 541-551).

[0070] Assays may also be designed to screen for compounds that regulateTRP8 expression at either the transcriptional or translational level. Inone embodiment, DNA encoding a reporter molecule can be linked to aregulatory element of the TRP8 gene and used in appropriate intactcells, cell extracts or lysates to identify compounds that modulate TRP8gene expression. Appropriate cells or cell extracts are prepared fromany cell type that normally expresses the TRP8 gene, thereby ensuringthat the cell extracts contain the transcription factors required for invitro or in vivo transcription. The screen can be used to identifycompounds that modulate the expression of the reporter construct. Insuch screens, the level of reporter gene expression is determined in thepresence of the test compound and compared to the level of expression inthe absence of the test compound.

[0071] To identify compounds that regulate TRP8 translation, cells or invitro cell lysates containing TRP8 transcripts may be tested formodulation of TRP8 mRNA translation. To assay for inhibitors of TRP8translation, test compounds are assayed for their ability to modulatethe translation of TRP8 mRNA in in vitro translation extracts.

[0072] In addition, compounds that regulate TRP8 activity may beidentified using animal models. Behavioral, physiological, orbiochemical methods may be used to determine whether TRP8 activation hasoccurred. Behavioral and physiological methods may be practiced in vivo.As an example of a behavioral measurement, the tendency of a test animalto voluntarily ingest a composition comprising the bitter tastant, inthe presence or absence of test inhibitor, may be measured. If thebitter tastant activates TRP8 in the animal, the animal may be expectedto experience a bitter taste, which would discourage it from ingestingmore of the composition. If the animal is given a choice of whether toconsume a composition containing bitter tastant only (with activatedTRP8) or a composition containing bitter tastant together with abitterness inhibitor (with lower levels of activated TRP8), it would beexpected to prefer to consume the composition containing the bitternessinhibitor. Thus, the relative preference demonstrated by the animalinversely correlates with the activation of the TRP8 channel.

[0073] Physiological methods include nerve response studies, which maybe performed using a nerve operably joined to a taste receptor cellcontaining tissue, in vivo or in vitro. Since exposure to bitter tastantwhich results in TRP8 activation may result in an action potential intaste receptor cells that is then propagated through a peripheral nerve,measuring a nerve response to a bitter tastant is, inter alia, anindirect measurement of TRP8 activation. An example of nerve responsestudies performed using the glossopharyngeal nerve are described inNinomiya, Y., et al., 1997, Am. J. Physiol. (London) 272:R1002-R1006.

[0074] The assays described above can identify compounds which modulateTRP8 activity. For example, compounds that affect TRP8 activity includebut are not limited to compounds that bind to the TRP8, and eitheractivate signal transduction (agonists) or block activation(antagonists). Compounds that affect TRP8 gene activity (by affectingTRP8 gene expression, including molecules, e, proteins or small organicmolecules, that affect transcription or interfere with splicing eventsso that expression of the full length or the truncated form of the TRP8can be modulated) can also be identified using the screens of theinvention. However, it should be noted that the assays described canalso identify compounds that modulate TRP8 signal transduction e.g.,compounds which affect downstream signaling events, such as inhibitorsor enhancers of G protein activities which participate in transducingthe signal activated by tastants binding to their receptor). Theidentification and use of such compounds which affect signaling eventsdownstream of TRP8 and thus modulate effects of TRP8 on the perceptionof taste are within the scope of the invention.

[0075] The compounds which may be screened in accordance with theinvention include, but are not limited to, small organic or inorganiccompounds, peptides, antibodies and fragments thereof, and other organiccompounds (e.g, peptidomimetics) that bind to TRP8 and either mimic theactivity triggered by the natural tastant ligand (i.e., agonists) orinhibit the activity triggered by the natural ligand (i.e.,antagonists).

[0076] Compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghten, R. et al., 1991, Nature 354:84-86); andcombinatorial chemistry-derived molecular library made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; (see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope bindingfragments thereof), and small organic or inorganic molecules.

[0077] Other compounds which may be screened in accordance with theinvention include but are not limited to small organic molecules thataffect the expression of the TRP8 gene or some other gene involved inthe TRP8 signal transduction pathway e.g., by interacting with theregulatory region or transcription factors involved in gene expression);or such compounds that affect the activity of the TRP8 or the activityof some other intracellular factor involved in the TRP8 signaltransduction pathway, such as, for example, a TRP8 associated G-protein.

5.6. COMPOSITIONS CONTAINING MODULATORS OF TRP8 AND THEIR USES

[0078] The present invention provides for methods of inhibiting a bittertaste resulting from contacting a taste tissue of a subject with abitter tastant, comprising administering to the subject an effectiveamount of a TRP8 inhibitor, such as a TRP8 inhibitor identified bymeasuring TRP8 activation as set forth in Section 5.5 supra. The presentinvention also provides for methods of inhibiting a bitter taste of acomposition, comprising incorporating, in the composition, an effectiveamount of a TRP8 inhibitor. An “effective amount” of the TRP8 inhibitoris an amount that subjectively decreases the perception of bitter tasteand/or that is associated with a detectable decrease in TRP8 activationas measured by one of the above assays.

[0079] The present invention further provides for a method of producingthe perception of a bitter taste by a subject, comprising administering,to the subject, a composition comprising a compound that activates TRP8activity such as a bitterness activator identified as set forth inSection 5.5 supra. The composition may comprise an amount of activatorthat is effective in producing a taste recognized as bitter by asubject.

[0080] Accordingly, the present invention provides for compositionscomprising bitterness activators and bitterness inhibitors. Suchcompositions include any substances which may come in contact with tastetissue of a subject, including but not limited to foods, beverages,pharmaceuticals, dental products, cosmetics, and wetable glues used forenvelopes and stamps.

[0081] In one set of embodiments, a bitterness inhibitor is used tocounteract the perception of bitterness associated with a co-presentbitter tastant. In these embodiments, a composition of the inventioncomprises a bitter tastant and a bitterness inhibitor, where thebitterness inhibitor is present at a concentration which inhibits bittertaste perception. For example, when the concentration of bitter tastantin the composition and the concentration of bitterness inhibitor in thecomposition are subjected to an assay as disclosed in Section 5.1 supra,the bitterness inhibitor inhibits the activation of TRP8 by the bittertastant.

[0082] The present invention may be used to improve the taste of foodsby decreasing or eliminating the aversive effects of bitter tastants. Ifa bitter tastant is a food preservative, the TRP8 inhibitors of theinvention may permit or facilitate its incorporation into foods, therebyimproving food safety. For foods administered as nutritionalsupplements, the incorporation of TRP8 inhibitors of the invention mayencourage ingestion, thereby enhancing the effectiveness of thesecompositions in providing nutrition or calories to a subject.

[0083] The TRP8 inhibitors of the invention may be incorporated intomedical and/or dental compositions. Certain compositions used indiagnostic procedures have an unpleasant taste, such as contrastmaterials and local oral anesthetics. The TRP8 inhibitors of theinvention may be used to improve the comfort of subjects undergoing suchprocedures by improving the taste of compositions. In addition, the TRP8inhibitors of the invention may be incorporated into pharmaceuticalcompositions, including tablets and liquids, to improve their flavor andimprove patient compliance (particularly where the patient is a child ora non-human animal).

[0084] The TRP8 inhibitors of the invention may be comprised incosmetics to improve their taste features. For example, but not by wayof limitation, the TRP8 inhibitors of the invention may be incorporatedinto face creams and lipsticks. In addition, the TRP8 inhibitors of theinvention may be incorporated into compositions that are not traditionalfoods, beverages, pharmaceuticals, or cosmetics, but which may contacttaste membranes. Examples include, but are not limited to, soaps,shampoos, toothpaste, denture adhesive, glue on the surfaces of stampsand envelopes, and toxic compositions used in pest control (e.g., rat orcockroach poison).

6. EXAMPLE CLONING AND CHARACTERIZATION OF THE TRP8 GENE

[0085] This following subsection describes the isolation andcharacterization of a transient receptor protein channel referred to asTRP8. The deduced amino acid sequence of TRP8 shows homology with otherTRP proteins. Northern Blot analysis indicates high level expression ofTRP8 RNA in taste receptor cells.

6.1. MATERIALS AND METHODS 6.1.1 CLONING OF THE TRP8 GENE

[0086] Single cell reverse transcription-polymerase chain reaction(RT-PCR) and differential screening were used to clone genesspecifically or selectively expressed in the subset of taste receptorcells that are positive for expression of the G protein gustducin.Individual gustducin-positive cells were isolated from mousecircumvallate papillae (Huang et al. 1999 Nature Neuroscience 2:1055-1062). The mRNAs from individual cells were reverse transcribedinto cDNA followed by PCR amplification. Multiple cDNA libraries fromsingle taste receptor cells were constructed by subcloning the amplifiedcDNAs into bacteriophage vectors. The cDNA libraries were analyzed bydifferential screening with self-probe (P³²-labelled amplified cDNAsfrom the same cell) and non-self probe (P³²-labeled amplified cDNAs fromanother taste cell). Hybridization was carried out at 65° C. for 20hours in 0.5 M sodium phosphate buffer (pH 7.3) containing 1% bovineserum albumin and 4% SDS. The membranes were washed twice at 65° C. in0.1% SDS, 0.5×SSC for 20 minutes and one time at 65° C. in 0.1% SDS,0.1×SSC for 15 minutes. The membranes were exposed to X-ray film at −80°C. with an intensifying screen for three days. Clones which morestrongly hybridize to self probe than to non-self probe were isolatedand their inserts sequenced.

[0087] Using this clone as a probe (LQSEQ91), a mouse taste tissue cDNAlibrary was screened for full-length clones under the same hybridizationconditions as specified above. Sequencing the clones containing thelongest inserts produced a full-length clone with greatest similarity toa family of calcium channel proteins known as transient receptorpotential (TRP) channels.

[0088] 25 μg of total RNA was isolated by acid guanidiniumthiocyanate/phenol/chloroform extraction (P. Chromczynski and N. Sacchi,1987, Anal. Biochem. 162:156) from the following mouse tissues: tastebud enriched epithelium, nontaste lingual epithelium, brain, retina,olfactory epithelium, stomach, small intestine, liver, spleen, kidney,lung, heart, thymus, uterus, testis and skeletal muscle. The RNAs wereelectrophoresed on 1.5% agarose gel containing 6.7% formaldehyde,transferred and fixed to a nylon membrane by UV irradiation. The blotwas hybridized with a radiolabeled 1.7 kb fragment generated from the3′-end of mouse TRP 8 cDNA by random priming with Exo(−) Klenowpolymerase in the presence of (∝-³²P)-dCTP. The hybridization wascarried out in 0.25 M sodium phosphate buffer (pH 7.2) containing 7% SDSat 65° C. with agitation for 24 hours. The membrane was washed twice in20 mM sodium phosphate buffer (pH 7.2) containing 5% SDS at 65° C. for40 minutes and twice in the same buffer containing 1% SDS at 65° C. for40 minutes. The blot was exposed to X-ray film at −80° C. with anintensifying screen for 5 days.

[0089] A BLAST search of human high throughput DNA sequences and genomicsequences was done using the mouse TRP8 sequence as the query. From thissearch a BAC clone was identified that contained the entire human TRP8gene. The Genscan program was then used to identify the predictedprotein-coding exons of the human TRP8 gene. The regions were alignedwith the mouse TRP8 cDNA to refine the predicted human TRP8 codingregion, leading to deduction of the entire human coding region.

6.1.2. NORTHERN HYBRIDIZATION

[0090] Total RNAs were isolated from several mouse tissues using theTrizol reagents, then 25 μg of each RNA was electrophoresed per lane ona 1.5% agarose gel containing 6.7% formaldehyde. The samples weretransferred and fixed to a nylon membrane by UV irradiation. The blotwas prehybridized at 65° C. in 0.25 M sodium phosphate buffer (pH 7.2)containing 7% SDS and 40 μg/ml herring sperm DNA with agitation for 5hours; hybridization for 20 hours with the ³²P-radiolabeled mouse TRP8probe was carried out in the same solution. The membrane was washedtwice at 65° C. in 20 mM sodium phosphate buffer (pH 7.2) containing 5%SDS for 40 minutes, twice at 65° C. in the same buffer containing 1% SDSfor 40 minutes, and once at 70° C. in 0.1×SSC and 0.1% SDS for 30minutes. The blot was exposed to X-ray film for 3 days at −80° C. withdual intensifying screens. The ³²P-labeled TRP8 probe was generated byrandom nonamer priming of a0.48 kb cDNA fragment of TRP8 correspondingto the 3′-UTR sequence using Exo(−) Klenow polymerase in the presence of(α-³²P)-dCTP.

6.1.3. IN SITU HYBRIDIZATION

[0091]³³P-labeled RNA probes [TRP8 (1.7 kb) and α-gustducin (1 kb)] wereused for in situ hybridization of frozen sections (10 μm) of mouselingual tissue. Hybridization and washing were as described(Asano-Miyoshi et al., 2000, Neurosci Lett 283:61-4). Slides were coatedwith Kodak NTB-2 nuclear track emulsion and exposed at 4° C. for 3 weeksand then developed and fixed.

6.1.4. IMMUNOCYTOCHEMISTRY

[0092] Polyclonal antisera against a keyhole limpethemocyanin-conjugated TRP8 peptide (aa 1028-1049) were raised inrabbits. The PLCβ2 antibody was obtained from Santa-CruzBiotechnologies; the anti-β-gustducin and anti-Gγ13 antibodies were asdescribed (Ruiz-Avila et al., 1995, Nature 376:80-5; Huang et al., 1999,Nat Neurosci 2:1055-62). Ten micron thick frozen sections of murinelingual tissue (previously fixed in 4% paraformaldehyde andcryoprotected in 20% sucrose) were blocked in 3% BSA, 0.3% Triton X-100,2% goat serum and 0.1% Na Azide in PBS for 1 hour at room temperatureand then incubated for 8 hours at 4° C. with purified antibody againstα-gustducin, or antiserum against TRP8 (1:800). The secondary antibodieswere Cy3-conjugated goat-anti-rabbit Ig for TRP8 andfluorescein-conjugated goat-anti-rabbit Ig for PLCβ2, α-gustducin orGγ13. TRP8 immunoreactivy was blocked by preincubation of the antiserawith the immunizing peptides at 20 μM. Preimmune serum did not show anyimmunoreactivity. Sections were double-immunostained with TRP8 plus oneof the following antibodies: anti-PLCβ2, anti-α-gustducin or anti-Gγ13.The sections were incubated sequentially with TRP8 antiserum,anti-rabbit-Ig-Cy3 conjugate, normal anti-rabbit-Ig, anti-PLCβ2 (oranti-α-gustducin or anti-Gγ13) antibody and finally withanti-rabbit-Ig-FITC conjugate with intermittent washes between eachstep. Control sections that were incubated with all of the above exceptanti-PLCβ2 (or anti-α-gustducin or anti-Gγ13) antibody did not show anyfluorescence in the green channel.

6.1.5. GENE EXPRESSION PROFILING

[0093] Single taste receptor cell RT-PCR products (5 μl) werefractionated by size on a 1.6% agarose gel and transferred onto a nylonmembrane. The expression patterns of the isolated cells were determinedby Southern hybridization with 3′-end cDNA probes for mouse TRP8,α-gustducin, Gβ3, Gγ13, PLCβ2 and G3PDH. Blots were exposed for fivehours at −80° C. Total RNAs from a single circumvallate papilla and asimilar-sized piece of non-gustatory epithelium were also isolated,reverse transcribed, amplified and analyzed as for the individual cells.

6.1.6. HETEROLOGOUS EXPRESSION

[0094] Oocytes were injected with 50 ng of TRP8 cRNA. 48 hours afterinjection oocytes were incubated in Thapsigargin (2 μM) and X-Rhod-1-AM(the Ca⁺⁺ sensing dye) for 3 hours at room temperature.

6.2. RESULTS 6.2.1. IDENTIFICATION OF A NOVEL TRP CHANNEL IN TASTE CELLS

[0095] Using single cell reverse transcription-polymerase chainreaction, a clone was isolated that was expressed in gustducin-positivecells but absent from gustducin-negative cells. A search of theexpressed sequence tag (EST) dbest database found no matches, suggestingthat this clone's pattern of expression is highly restricted to tissuesnot generally found in EST databases, such as taste tissue.

[0096] Using this clone as a probe, a mouse taste tissue cDNA librarywas screened for full-length clones. Sequencing the clones containingthe longest inserts produced a full-length clone with the sequenceindicated in FIG. 1. The deduced amino acid sequence of the cDNA cloneis shown in FIG. 2.

[0097] The isolated cDNA showed the greatest similarity to a family ofcalcium channel proteins known as transient receptor potential (TRP)channels. The similarity of the isolated clone to this family ofproteins indicated that a TRP channel had been identified. Currentlyseven TRP channels are known to exist, making this clone the eighthmember, named by convention TRP8. Mouse TRP8 (TRP8) is most closelyrelated to TRP7 with an identity at the amino acid level of 40%. Thepredicted topography of the TRP8 channel inserted within the cellmembrane is presented in FIGS. 6A-C.

[0098] Based upon homology of the mouse clone with a region of humanchromosome 11p15.5 contained in a BAC clone (genebank #AC003693) a humanTRP8 ortholog was identified. The nucleotide sequence of the human TRP8gene, as well as the deduced amino acid sequence, are depicted in FIGS.3A-B and 4, respectively. A comparison of the murine and human TRP 8proteins is shown in FIG. 5. This region of human chromosome 11p15.5 issyntenic with the distal region of mouse chromosome 7. In both cases,TRP8 and hTRP8 map between genes for Kvlqt1 and TSSC4.

6.2.2. TRP8 IS SELECTIVELY EXPRESSED IN TASTE TISSUE

[0099] Although TRP8 was identified during a differential screen ofα-gustducin-positive (+) vs. α-gustducin (−)⁻ TRCs, it was possible thatTRP8 might be more broadly expressed in other taste cells and/ortissues. To determine the tissue distribution of TRP8 MRNA a northernblot with multiple murine tissues was carried out. An TRP8 3′-UTR probehybridized predominantly to a transcript of 4.5 kb in taste tissue, withno detectable expression in control non-taste tissue. Moderateexpression was detected in stomach and small intestine; weak expressionwas noted in uterus and testis (FIG. 7A). This is in contrast to theresults of Enklaar et al., (2000, Genomics 67:179-87). Using anRT-PCR-generated probe designed to amplify the 3′ portion of TRP8'scoding region they detected highest expression in liver and low levelexpression in other peripheral tissues (e.g. heart, brain, kidney andtestis). Their RT-PCR probe may have detected by cross-hybridizationother TRP8 mRNAs or an alternatively spliced MRNA with a different3′-end from that present in our 3′-UTR probe. As an independent measureof expression of TRP8, we carried out western blots using an anti-TRP8antibody (FIG. 7B). TRP8 protein of the predicted molecular weight (˜130kDa) was detected in stomach and small intestine; a species of higherthan expected molecular weight was identified in liver and kidney andmay represent either an TRP8 -related protein or an TRP8 product from analternatively spliced message.

6.2.3. TRP8 IS EXPRESSED IN PARTICULAR SUBSETS OF TASTE RECEPTOR CELLS

[0100] In situ hybridization was used to determine the cellular patternof expression of TRP8 in mouse TRCs. TRP8 mRNA was observed in TRCs incircumvallate and foliate papillae, but not in the surroundingnon-gustatory epithelia (FIG. 8). TRP8⁺ TRCs were present in themajority of the taste buds, although not all TRCs were positive,suggesting restricted expression to a subset of TRCs. The generalpattern of TRP8 expression was comparable to that of α-gustducin,although the α-gustducin signal was somewhat more intense (FIG. 8D).Controls with sense probes showed minimal non-specific hybridization totaste tissue with either the TRP8 probe (FIG. 8B) or the (α-gustducinprobe (FIG. 8E).

[0101] To determine if TRP8 is co-expressed in TRCs with signaltransduction elements that might be involved in its activation, weperformed single and double immunohistochemistry of TRC-containingtissue sections. TRP8 protein was co-expressed absolutely with Gγ13(FIG. 9ABC) and PLCβ2 (FIG. 9GHI), suggesting that these three moleculesmight be part of a common signal transduction pathway. TRP8 co-expressedlargely, but not absolutely, with (α-gustducin (FIG. 9DEF): a subset ofthe TRP8⁺ TRCs were negative for α-gustducin, although all α-gus⁺ TRCswere positive for TRP8. This pattern is consistent with our observationsthat α-gus⁺ TRCs constitute a subset of TRCs that are positive for Gγ13,Gβ1, PLCβ2 and IP₃R3 (Huang et al, 1999,). The slight differences indistribution at the cellular level among the different molecules couldbe explained by the different topologies that each protein displays:TRP8 is an integral membrane protein, whereas α-gustducin and PLCβ2 aremembrane-associated proteins. The expression of human TRP8 (hTRP8 ) inhuman fungiform taste buds was also confirmed.

[0102] To independently monitor co-expression of TRP8 in TRCs with theabove-mentioned signal transduction elements, as well as with Gβ1 andGβ3, a single cell expression profiling was carried out (Huang et al.,1999, Nat Neurosci 2:1055-62)). In this way it was determined thatexpression of α-gustducin, Gβ1, Gβ3, Gγ13, PLCβ2 and TRP8 was restrictedto taste tissue (FIG. 10, left panel), and that in this set of 24 TRCs,TRP8 co-expressed absolutely with α-gustducin, Gβ3, Gγ13, PLCβ2 (FIG.10, right panel); expression of TRP8 also overlapped in large part withthat of Gβ1 (15 of 19 TRP8⁺ cells were also Gβ1⁺). The coincidentexpression of these various signal transduction molecules with TRP8could provide the physical opportunity for activation of TRP8 by IP₃ (byactivation of IP₃ receptors) or DAG (by direct activation of TRP8)generated by a signaling pathway in which GPCRs coupled toheterotrimeric gustducin (i.e. α-gustducin/β3/γ13) or to otherGα/β1,β3/γ13-containing heterotrimers might release βγ to activatePLCβ2. Consistent with this is the recent identification in TRCs of IP₃receptor subtype III (IP₃R3), and the demonstration that IP₃R3co-localizes in large part with α-gustducin, Gγ13 and PLCβ2.

6.2.4. OTHER TRP FAMILY MEMBERS ARE NOT DETECTABLY EXPRESSED IN TASTETISSUE

[0103] Native TRP channels are thought to form homo- andhetero-multimers. To identify potential partners for TRP8 in TRCs PCRwas used to determine if murine TRP channels 1-6 (TRP 1-6) are expressedin taste tissue (brain tissue provided a positive control).Amplification by the PCR using primer pairs specific for TRP 1-6identified products of the correct size for all six TRP family memberswhen brain cDNA was used as the template (FIG. 11, lower panel); DNAsequencing of these products confirmed amplification of all six TRPfamily members. TRP8 was not amplified when brain cDNA was the template(FIG. 11, lower panel), although it was amplified when taste cDNAprovided the template (FIG. 11, upper panel) (amplification of TRP8 wasconfirmed by DNA sequencing). None of the other six TRP family memberswere amplified when taste tissue cDNA was used as the template (FIG. 11,upper panel), suggesting that they are not highly expressed, if at all,in TRCs. In a separate experiment using TRP7 specific primers, TRP7 wasdetected by PCr in brain cDNA, but not in taste cDNA. Novel TRP channelsbeyond these seven members might be expressed in TRCs, but at thepresent time it would appear that TRP8 is the only known TRP channelhighly expressed in taste tissue, and as shown above, in TRCs.

6.2.5. EXPRESSED TRP8 ACTS AS A STORE OPERATED CHANNEL

[0104] To determine if TRP8 can function as a calcium channel, TRP8 wasexpressed in Xenopus oocytes. The oocytes possess an endogenouscalcium-activated chloride conductance (ICl_(Ca)) that may be used tomonitor Ca⁺⁺ influx due to activation of store operated Ca⁺⁺ channelsbelonging to the TRP family. TRP8 RNA obtained by in vitro transcriptionwas injected into Xenopus oocytes and two electrode voltage clamprecordings were performed two days later. To induce depletion ofinternal Ca⁺⁺ stores, oocytes were incubated for 2 hours before therecording in 2 μM thapsigargin (TPN), an irreversible inhibitor of thesarco(endo)plasmic reticulum Ca⁺⁺-ATPase (SERCA).

[0105] Representative recording traces of oocytes injected with TRP8 RNAand treated with TPN demonstrated a robust and distinct inward currentelicited by the addition of Ca⁺⁺ to the external bath (FIG. 12A). Thesetraces differ dramatically from those of control oocytes injected withwater (FIG. 12B), indicating that TRP8 encodes a functional Ca⁺⁺ channelwhose activation is dependent on the filling status of the internal Ca⁺⁺stores (compare FIG. 12 panels A and B), and whose function relies onthe availability of external Ca⁺⁺. The control oocytes express anendogenous TRP channel (XTrp) (Bobanovic et al., 1999, Biochem J.340:593-9)) that can be activated by TPN treatment (FIG. 12B). Analysisof the total inward current (FIG. 12D) generated under conditions whenCa⁺⁺ is present in the extracellular medium clearly demonstrated theeffect of TRP8 expression. To confirm that TRP8 protein was actuallyexpressed in the oocytes, we carried out a western blot of the membraneproteins from TRP8 RNA-injected oocytes using an anti-TRP8 antibody: a130 kDa protein of the expected size was detected.

[0106] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. Various references are cited herein, thedisclosures of which are incorporated by reference in their entireties.

We claim:
 1. An isolated nucleic acid molecule comprising a nucleotidesequence that encodes the amino acid sequence shown in FIG.
 2. 2. Theisolated nucleic acid molecule of claim 1 comprising the DNA sequence ofFIG.
 1. 3. An isolated nucleic acid molecule comprising the DNA sequenceof FIG.
 3. 4. The isolated nucleic acid molecule of claim 3 comprising anucleotide sequence that encodes the amino acid sequence shown in FIG.4.
 5. An isolated nucleic acid molecule comprising a nucleotide sequencethat hybridizes to the nucleotide sequence of claim 1 or 3 understringent conditions and encodes a functionally equivalent gene product.6. An isolated nucleic acid molecule comprising a nucleotide sequencethat hybridizes to the nucleic acid of claim 1 or 3 under moderatelystringent conditions and encodes a functionally equivalent TRP8 geneproduct.
 7. An isolated nucleic acid molecule that is a TRP8 antisensemolecule.
 8. An isolated polypeptide comprising the amino acid sequenceof FIG.
 2. 9. An isolated polypeptide comprising the amino acid sequenceof FIG.
 4. 10. An isolated polypeptide comprising the amino acidsequence encoded by a nucleotide sequence that hybridizes to thenucleotide sequence of claim 1 or 3 under stringent conditions andencodes a functionally equivalent gene product.
 11. An isolatedpolypeptide comprising the amino acid sequence encoded by a nucleotidesequence that hybridizes to the nucleotide sequence of claim 1 or 3under moderately stringent conditions and encodes a functionallyequivalent gene product.
 12. A purified fragment of a TRP8 proteincomprising a domain of the TRP8 protein selected from the groupconsisting of the transmembrane domain and cytoplasmic domain.
 13. Achimeric protein comprising a fragment of a TRP8 protein consisting ofat least 6 amino acids fused via a covalent bond to an amino acidsequence of a second protein, in which the second protein is not a TRP8protein.
 14. An antibody which is capable of binding a TRP8 protein. 15.A recombinant cell containing the nucleic acid of claim 5 or
 6. 16. Amethod of producing a TRP8 protein comprising growing a recombinant cellcontaining the nucleic acid of claim 5 or 6 such that the encoded TRP8protein is expressed by the cell, and recovering the expressed TRP8protein.
 17. A method for identifying a compound that induces theperception of a bitter taste comprising: (i) contacting a cellexpressing the TRP8 channel protein with a test compound and measuringthe level of TRP8 activation; (ii) in a separate experiment, contactinga cell expressing the TRP8 channel protein with a vehicle control andmeasuring the level of TRP8 activation where the conditions areessentially the same as in part (i); and (iii) comparing the level ofactivation of TRP8 measured in part (i) with the level of activation ofTRP8 in part (ii), wherein an increased level of activated TRP8 in thepresence of the test compound indicates that the test compound is a TRP8inducer.
 18. A method for identifying a compound that inhibits theperception of a bitter taste and/or promotes the perception of a sweettaste comprising: (i) contacting a cell expressing the TRP8 channelprotein with a test compound in the presence of a bitter tastant andmeasuring the level of TRP8 activation; (ii) in a separate experiment,contacting a cell expressing the TRP8 channel protein with a bittertastant and measuring the level of TRP8 activation, where the conditionsare essentially the same as in part (i); and (iii) comparing the levelof activation of TRP8 measured in part (i) with the level of activationof TRP8 in part (ii), wherein a decrease level of activation of TRP8 inthe presence of the test compound indicates that the test compound is aTRP8 inhibitor.
 19. A method for identifying an inhibitor of bittertaste in vivo comprising: (i) offering a test animal the choice ofconsuming either (a) a composition comprising a bitter tastant or (b)the composition comprising the bitter tastant as well as a testinhibitor; and (ii) comparing the amount of consumption of thecomposition according to (a) or (b), wherein greater consumption of thecomposition according to (b) has a positive correlation with an abilityof the test inhibitor to inhibit the perception of bitter tasteassociated with the tastant.
 20. A method for identifying an activatorof bitter taste in vivo comprising: (i) offering a test animal thechoice of consuming either (a) a control composition or (b) thecomposition comprising a test activator; and (ii) comparing the amountof consumption of the composition according to (a) or (b), whereingreater consumption of the composition according to (a) has a positivecorrelation with an ability of the test activator to activate theperception of bitter taste.
 21. A method of inhibiting a bitter tasteresulting from contacting a taste tissue of a subject with a bittertastant, comprising administering to the subject an effective amount ofa bitterness inhibitor.
 22. A method of producing the perception of asweet taste by a subject, comprising administering, to the subject, acomposition comprising a compound that acts as a bitterness inhibitor inaddition to eliciting a sweet taste.
 23. A method of producing theperception of a bitter taste by a subject, comprising administering, tothe subject, a composition comprising a compound that acts as abitterness activator.